To align mass spectrometry imaging (MSI) workflows with the realities of clinical pathology, we develop applications that make formalin-fixed paraffin-embedded (FFPE) tissues accessible for MSI using cutting-edge high-resolution (HR) instrumentation. As such we lower the threshold for the adaptation of MSI in the field of pathology.
Two types of atmospheric pressure ionization HRMS are employed: orbitrap (ThermoFisher) instruments interfaced with an AP/MALDI UHR (MassTech) source on DHB coated deparaffinized samples, and DESI MRT (Waters). FFPE samples procured from the university hospital pathology biobank are sectioned at 5 μm thickness (microtome) on standard glass microscope slides.
Using optimized antibody- and enzyme-free protocols we successfully image a variety of physiologically relevant biomolecules, including endogenous neuropeptides and metabolites, directly from FFPE tissue sections. This capability, designated mass spectrometry histochemistry (MSHC), mirrors the workflow of immunohistochemistry (IHC), improves molecular specificity, offers a spatial resolution down to 5×5 μm² and can be applied directly to the millions of FFPE tissue blocks stored in biobanks worldwide. We illustrate this showing MALDI (using DHB as matrix) and (matrix-free) DESI high-resolution MSHC data on different FFPE tissue samples.
The potential of MSHC is huge; we can now retrospectively analyze archived samples from pathology biobanks to discover small molecule biomarkers linked to the seriousness of the disease, how patients respond(ed) to treatment, or their long-term outlook. Since MSHC works directly on the same slides already used for routine pathology, it fits easily into existing lab workflows – making it much simpler to bring into clinical practice. This also facilitates integration of MSHC data with other pathology imaging modalities currently in practice on FFPE samples, including IHC, H&E.
MSHC is an elegant way to generate an extra dimension of data which can be assimilated with the complete pathology knowledgebase well-documented in biobanked disease archives. Even without molecular identification of MSHC ion signals, we predict that AI will soon help to link MSHC data to relevant clinical decisions, similar to microbial identifications confidently based on MALDI profiles.
The promise of MSHC is no longer theoretical. The tools exist, the protocols are feasible, and samples are waiting. MSHC transforms existing pathology archives into living resources for discovery and diagnosis, offering the potential to revolutionize how we classify disease and tailor treatment. What’s needed now is collaborative engagement from the pathology community – to help select cases, validate findings, regulate protocols and integrate this new layer of molecular insight into everyday diagnostics.